Reverse transcription is the process by which a retrovirus such as HIV-1 converts its genetic material (single-stranded ss RNA) into a double-stranded DNA copy that is integrated into host chromosomal DNA. This process is complex and is catalyzed by the virion-associated enzyme, reverse transcriptase (RT). However, another viral protein, the nucleocapsid protein (NC), is also required for efficient and specific viral DNA synthesis. (A) We study the mechanistic basis for NC activity. HIV-1 NC is a small, basic nucleic acid binding protein with two zinc fingers, each containing the invariant CCHC zinc-coordinating motifs. It is a nucleic acid chaperone, i.e., NC has the ability to catalyze conformational rearrangements that result in formation of the most thermodynamically stable nucleic acid structures. This property is critical for promoting the two strand transfer events that are needed for synthesis of full-length plus- and minus-strand viral DNA. In minus-strand transfer, the initial product of reverse transcription, (-) strong stop DNA, is translocated to the 3-prime end of viral RNA (termed acceptor RNA) in a reaction facilitated by base-pairing of the complementary repeat regions, which are present at the ends of the RNA and DNA partners. (i) Recent studies have focused on the nucleic acid chaperone activity of HIV-1 NC when it is embedded in Gag (the precursor to the viral structural proteins) and partially processed Gag proteins. Activity was assayed using a reconstituted minus-strand transfer assay system, a highly sensitive readout for chaperone function. Our experiments demonstrated that chaperone activity resides in the NC domain of the precursor proteins. Moreover, we have reported for the first time that Gag and Gag-derived proteins have annealing and helix destabilizing activity, since these proteins stimulate minus-strand transfer. Surprisingly, unlike NC, high concentrations of Gag proteins block the DNA elongation step in strand transfer. This result is consistent with nucleic acid-driven multimerization of Gag and the slow dissociation of Gag from bound nucleic acid, which prevent RT from traversing the template (roadblock mechanism). These findings illustrate one reason why NC (and not Gag) has evolved as critical cofactor in reverse transcription. Additionally, the ability of Gag to act as a roadblock to DNA polymerization could help to prevent premature viral DNA synthesis. We are currently investigating the effect of zinc finger mutations in the NC domain of Gag on minus-strand transfer as well as on RT-catalyzed elongation of tRNA(Lys3) that is annealed to viral RNA in the presence of Gag. (ii) During synthesis of (-) strong-stop DNA, the RNase H activity of RT degrades the viral RNA template. As RT reaches the end of the template, small 5-prime RNA fragments remain annealed to the DNA, since RNase H cleavage of blunt-ended substrates is inefficient. However, these fragments must be removed so that minus-strand transfer can proceed. We hypothesized that fragment removal could be facilitated by NC destabilization of the short duplex and/or RNase H cleavage. To test this hypothesis, a small RNA complementary to the 3-prime end of (-) strong-stop DNA was heat annealed to the DNA. Additional components were added (e.g., acceptor RNA, NC, RT) and following incubation, the percent transfer product synthesized was determined. Since strand transfer cannot occur unless the RNA is removed, this assay uses formation of the transfer product as the readout for RNA removal. We also measured the efficiency of fragment removal in the absence of reverse transcription using gel-shift mobility and FRET assays. Collectively, our results show that 5-prime terminal RNA fragments can be removed by NC alone, but RNase H cleavage is required for maximal removal. The ability of NC to coordinate zinc is also needed for this activity. These findings are in excellent agreement with our earlier studies of the tRNA removal step in plus-strand transfer (Wu et al. J.Virol. 73:4794-4805, 1999) and the ability of NC to block mispriming during initiation of plus-strand DNA synthesis (Post et al. NAR 37:1755-1766, 2009). (B) Our interest in host proteins that might affect HIV-1 reverse transcription and replication has led us to investigate human APOBEC3 (A3) proteins, a family of seven cytidine deaminases, which also have antiviral activity. Our initial studies focused on A3G, which blocks HIV-1 reverse transcription in the absence of the viral protein Vif (virus infectivity factor). Currently, we are studying the human A3A protein, which functions as a cell defense factor by strongly inhibiting retrotransposition of LINE-1 elements, a class of mobile genetic elements. These elements are potentially detrimental to the human genome, since they can insert into coding regions of functional genes. To test the role of A3A deaminase activity in the inhibition of LINE-1 retrotransposition, we performed cell-based assays with A3A mutants and correlated the levels of retrotransposition with catalytic activity. Structure-guided mutagenesis, based on A3A structural homology to the known crystal structure of the A3G catalytic domain, was used to design the mutants. In general, mutants defective in deamination were also found to exhibit less anti-retrotransposition activity, suggesting a correlation between these two activities. To obtain further insights into the molecular properties of A3A, we are using highly purified recombinant protein to study the kinetics of cytidine deamination and binding to nucleic acids. A3A is a potent deaminase and with equivalent amounts of enzyme and ssDNA substrate, deamination is complete within 5 min. A3A binds ssRNA more efficiently than ssDNA, which is surprising, since only ssDNA is a substrate for the enzyme. Further work with purified A3A is in progress to probe additional aspects of its biochemical activities. (C) Our laboratory has also been investigating the role of the HIV-1 capsid protein (CA) in early postentry events, a stage in the infectious process that is still not completely understood. In current work, we are studying the function of the flexible peptide, known as the interdomain linker, which connects the N- and C-terminal domains of CA. We made single alanine or leucine substitutions in each of the five linker residues (CA residues 146-150) and in the two flanking residues. Although all of the mutants produce virus particles, only two mutants (S146A and T148A) have infectivity in a single-cycle replication assay. The lack of infectivity of three other mutants (Y145A, I150A, and L151A) is correlated with defects in core morphology and stability, inability of purified CA proteins to assemble into a recognizable structure in vitro, as well as abrogation of viral DNA synthesis. Interestingly, two linker mutants (P147L and S149A), while poorly infectious, display an attenuated phenotype and surprisingly, their infectivity is rescued when env-minus virions are pseudotyped with the vesicular stomatitis envelope glycoprotein. Moreover, despite having unstable cores, these mutants (i) assemble a significant number of viral cores with seemingly normal architecture, (ii) assemble tubular structures in vitro that resemble wild-type assemblies, and (iii) synthesize viral DNA in infected cells, although less efficiently than wild-type virus. These results clearly demonstrate that the interdomain linker region has a critical role in facilitating proper core assembly and stability, which in turn ultimately impacts the ability of the virus to undergo efficient replication.